Nursery‐linked plantation outbreaks and evidence for multiple introductions of the pitch canker pathogen Fusarium circinatum into South Africa

In recent years, Pinus plantation forestry has been significantly hampered by outbreaks of pitch canker caused by the fungus Fusarium circinatum. This study investigated the role of Pinus host, geographic origin and reproductive mode in structuring the F. circinatum populations in plantations. For this purpose, 159 isolates originating from diseased plantation trees in the Western and Eastern Cape Provinces of South Africa were genotyped using 10 microsatellite markers. Analyses of these data revealed 30 multilocus haplotypes and that the populations were distinct based on geographic origin as well as host. However, shared haplotypes were observed between populations, showing that these populations are connected, possibly through the movement of haplotypes. A second aim was to determine whether the genetic variation found in these populations of the fungus could be attributed to outbreaks of the seedling disease caused by this pathogen in Pinus nurseries. To achieve this goal, an additional set of 43 isolates originating from pine seedling nurseries was genotyped and analysed. The results showed that the populations of F. circinatum in plantations most probably originated from the nursery outbreaks that occurred prior to the plantation outbreak. Inferences regarding reproductive mode further showed that sexual reproduction has little impact on the genetic makeup of the F. circinatum populations and that they primarily reproduce asexually. Overall, the results of this study showed that the F. circinatum diversity in South Africa has arisen due to multiple introductions of the pathogen and is not due to sexual reproduction.     

Inheritance of virulence in Fusarium circinatum,the cause of pitch canker in pines

Wildtype strains of Fusarium circinatum, the causal agent of pitch canker, were crossed to obtain an F₁ generation. Progeny of this cross were tested for virulence by inoculating Pinus radiata seedlings, and were found to induce a wide range of lesion lengths. Two strains from the F₁ generation that induced long lesions (= high virulence) were used as parents to produce an F₂ generation, followed by a second round of selection for high virulence to obtain an F₃ generation. Mean lesion lengths were not significantly different between the three generations (P ≥ 0.196). A parallel set of crosses was performed to select for low virulence by using progeny in the F₁ and F₂ generations that induced short lesions as parents for F₂ and F₃ generations, respectively. In this case, both rounds of selection resulted in a significant reduction in mean lesion length, from 33.8 ± 0.8 mm in the F₁ generation, to 19.7 ± 0.7 and 12.9 ± 0.7 mm in the F₂ and F₃ generations, respectively. Thus it is apparent that F. circinatum retains the genetic capacity for avirulence to pines, which could reflect a lack of strong selection for virulence in nature. Progeny of a cross between high and low virulence parents manifested nearly continuous variation in lesion lengths, consistent with virulence being a quantitatively inherited trait. Based on this cross, broad‐sense heritability (H²) was determined to be 0.74, which suggests that virulence is under strong genetic control.     

Alternative species to replace Monterey pine plantations affected by pitch canker caused by Fusarium circinatum in northern Spain

The pitch canker pathogen Fusarium circinatum was first found to cause damage in nurseries and pine plantations in northern Spain in 2004. Since then, establishment of pine plantations in the region has decreased as a result of the prohibitions placed on planting Pinus spp. and Pseudotsuga menziesii in areas affected by the disease. However, although most pine species have been found to be susceptible to the pathogen under nursery conditions, little is known about how the fungus affects the trees in the field. Furthermore, it is not known whether some of the native or exotic species commonly planted in the area are also susceptible to F. circinatum. The aim of this study was to evaluate the susceptibility of several conifer species commonly planted in northern Spain to the pitch canker pathogen. For this purpose, two different trials were carried out, one under controlled laboratory conditions and the other in the field. Although most of the conifers were affected by the pathogen in the laboratory tests, only Pinus radiata, Pinus nigra, Pinus pinaster and Pinus uncinata were susceptible to the pathogen in the field.     

Unique clones of the pitch canker fungus, <Emphasis Type="Italic">Fusarium circinatum,</Emphasis> associated with a new disease outbreak in South Africa

Pitch canker of pines is caused by the fungus Fusarium circinatum. In South Africa, this pathogen has mostly been a nursery problem. From 2005, however, outbreaks of pitch canker have been reported from established Pinus radiata and P. greggii in the Western and Eastern Cape Provinces. Most recently, pitch canker-like symptoms were observed on 10-year-old P. greggii trees in a plantation in the midlands of the KwaZulu-Natal (KZN) Province. The aim of this study was to: (i) identify the causal agent of the observed symptoms, (ii) determine the genetic diversity, and (iii) the mode of reproduction of this fungal population. Furthermore, the aggressiveness of isolates from these trees was compared with that of isolates obtained previously from P. patula in South Africa. Isolates from the P. greggii trees in KZN were confirmed as F. circinatum based on both morphology and DNA sequence analyses. Microsatellite marker analyses revealed the presence of five genotypes of F. circinatum, not previously reported from other plantations in South Africa, with one of these genotypes being dominant. These genotypes were all pathogenic to P. patula and P. elliottii. No evidence of sexual reproduction was detected in the KZN population of the fungus. This was consistent with the fact that isolates from P. greggii were all of the MAT-2 mating type, in contrast to previously collected isolates from across South Africa that included both mating types. The results suggest that the outbreak of pitch canker on P. greggii in KZN represents a separate introduction of F. circinatum into the region with important implications for managing the disease.     

Genotypic diversity in a South African population of the pitch canker fungus Fusarium subglutinans f.sp. pini

The genotypic diversity in a South African population of Fusarium subglutinans f.sp. pini ( F.s. pini ) was determined, based on the number of vegetative compatibility groups (VCGs). Isolates of F.s. pini from South Africa (69), California (five) and Florida (19) were included in the study. The nit1 (or nit3 ) and NitM mutants were selected as chlorate resistant sectors and paired on minimal medium. The South African isolates of F.s. pini were assigned to 23 different VCGs. No heterokaryons formed between isolates from South Africa, California and Florida. The high degree of genotypic diversity in the South African population of F.s. pini is probably due to some level of sexual reproduction in the population.     

Fusarium species associated with stalk rot and head blight of grain sorghum in Queensland and New South Wales,Australia

Historical records report Fusarium moniliforme sensu lato as the pathogen responsible for Fusarium diseases of sorghum; however, recent phylogenetic analysis has separated this complex into more than 25 species. During this study, surveys were undertaken in three major sorghum‐producing regions in eastern Australia to assess the diversity and frequency of Fusarium species associated with stalk rot‐ and head blight‐infected plants. A total of 523 isolates were collected from northern New South Wales, southern Queensland and central Queensland. Nine Fusarium species were isolated from diseased plants. Pathogenicity tests confirmed F. andiyazi and F. thapsinum were the dominant stalk rot pathogens, whilst F. thapsinum and species within the F. incarnatum–F. equiseti species complex were most frequently associated with head blight.     

Susceptibility of pines in South Africa to the pitch canker fungus subglutinans f.sp. pini

Fusarium subglutinans f.sp. pini (F.s. pini)is the causal agent of pitch canker of pines. The fungus has recently been found in South Africa on the diseased roots of seedlings, but has as yet not been detected on mature trees in commercial forests. Inoculation of 1 -year-old and seedlings with isolates of resulted in canker development and shoot mortality. No significant differences in virulence were found among eight isolates of the pathogen on and but isolate MRC 6214 was significantly more virulent on seedlings than MRC 6209. Disease development was significantly more severe on and than on Pathogenicity tests on 4-year-old and trees yielded comparable results. Resinous cankers, similar to those described for pitch canker, developed on trees in the vicinity of inoculation points but development ceased before stems were girdled.     

Stem rot of jewel orchids caused by a new forma specialis,Fusarium oxysporum f. sp. anoectochili in Taiwan

Stem rot of Anoectochilus formosanus (Af) caused by Fusarium oxysporum (Fo) is a major limiting factor to jewel orchid production in Taiwan. Fo causes discoloration in vascular tissues. However, some newly collected Fo isolates from Af stem rot do not cause vascular discoloration, suggesting changes may have occurred in the pathogen. Among recent Fo isolates from Af there are two colony types, the cottony alba (CA) and the sporodochial (S). In order to confirm that both colony types cause Af stem rot, 200 isolates were obtained from diseased stems in Nantou County and characterized by colony type and whether or not the infected plants had vascular discoloration. Isolates of both the CA and S types caused stem rot of Af; some isolates in each colony type caused vascular discoloration whilst others did not. Pathogenicity tests with 22 isolates resulted in stem rot disease severity ratings on Af of 3·1–4·0 and 2·1–4·0 with CA and S type colonies, respectively. The same isolates failed to cause disease on Cattleya, Dendrobium or Phalaenopsis plants. Phylogenetic analysis of partial intergenic spacer sequences showed that these isolates were distinguishable from other formae speciales of Fo and could be separated into two groups correlated with the CA or S type colonies with high bootstrap. Based on pathogenic, morphological and molecular characterizations, the Fo that causes stem rot of Af is proposed to be a new forma specialis, F. oxysporum f. sp. anoectochili, with different pathotypes.     

Pathogenicity of Fusarium graminearum and F. meridionale on soybean pod blight and trichothecene accumulation

Soybean (Glycine max) is the most important crop in Argentina. At present Fusarium graminearum is recognized as a primary pathogen of soybean in several countries in the Americas, mainly causing seed and root rot and pre‐ and post‐emergence damping off. However, no information about infections at later growth stages of soybean development and pathogenicity of F. graminearum species complex is available. Therefore, the objectives of this study were to compare the pathogenicity of F. graminearum and F. meridionale isolates towards soybean under field conditions and to evaluate the degree of pathogenicity and trichothecene production of these two phylogenetic species that express different chemotypes. Six isolates of F. graminearum and F. meridionale were evaluated during 2012/13 and 2013/14 soybean growing seasons for pod blight severity, percentage of seed infected in pods and kernel weight reduction. The results showed a higher aggressiveness of both F. graminearum and F. meridionale species during the 2013/14 season. However, the differences in pathogenicity observed between the seasons were not reflected in a distinct trichothecene concentration in soybean seeds at maturity. Fusarium meridionale isolates showed similar pathogenicity to F. graminearum isolates but they were not able to produce this toxin in planta during the two field trials.     

High diversity and clonality are hallmarks of Fusarium circinatum in South Africa

Pitch canker on plantation-grown Pinus species, caused by the fungal pathogen Fusarium circinatum, first appeared in the western and southern Cape regions of South Africa. However, outbreaks have subsequently been reported from the major plantation growing regions of KwaZulu-Natal and Limpopo in the eastern, summer rainfall regions of the country. It is more than 10 years since the last detailed population genetics studies on F. circinatum in the region were conducted. To shed light on the population biology of F. circinatum in this region of South Africa, we used microsatellite markers and mating-type assays to study a collection of 296 isolates from different nurseries and plantation sites. Our results showed that populations in the region are highly diverse, but strongly interconnected, with various genotypes shared across nursery and plantation collection sites. In contrast to nursery populations, those associated with pitch canker outbreaks were characterized by the presence of relatively small numbers of dominant genotypes that were generally widespread across the region. Opposite mating-type individuals occurred in most of the isolate collections, but multilocus linkage disequilibrium analyses pointed towards clonality being the main reproductive mode of F. circinatum in the region. Most of the pathogen's genetic variation could probably have resulted from multiple different introductions into the country and more specifically, into the summer rainfall region. Because the spread and establishment of invasive pathogens are typically driven by aggressive clones, the results of this study provide important considerations for current and future Pinus disease management strategies.     

Fusarium mangiferae localization in planta during initiation and development of mango malformation disease

Mango malformation disease (MMD), caused by Fusarium mangiferae, is a major constraint to mango production, causing significant yield reduction resulting in severe economic impact. The present study characterizes fungal localization in planta during initiation and development of vegetative and floral malformation. Young mango trees were artificially inoculated with a green fluorescent protein (GFP)‐expressing strain of F. mangiferae. Shoots and buds were sampled periodically over a period of more than a year and localization of the GFP‐expressing fungi was determined using confocal microscopy. Fungal localization appears to be epiphytic: mycelia remained in close contact with the plant surface but did not penetrate the tissue. In vegetative malformation and in young inflorescences, the fungus was confined to protected regions between scales, young leaf bases and buds. Fungal colonization was only very rarely detected on open leaves or on exposed shoot sections. In developed flowers, mycelia were localized mainly to protected regions at the base of the flower organs. Upon development of the inner flower organs, specific mycelial growth occurred around the anthers and the style. Mycelial penetration through the stylar tract into aborting carpels was observed. For several months, mycelia were confined to the surface of the organs and were not detected within plant tissues. Only at later stages, transient saprophytic growth of the fungus was detected causing the malformed inflorescences to senesce and collapse, concurrent with dispersion of conidia. Implications of the present study on MMD in natural field infections are discussed.     

Perspectives on the specific targeting of Fusarium graminearum for the development of alternative head blight treatment approaches

Diseases of agricultural crops caused by fungi have devastating economic and health effects. Fusarium head blight (FHB) is one of the most damaging diseases of wheat and other small grain cereals. FHB reduces agricultural yield while also affecting food supply and safety through deposition of toxins (mycotoxins/phytotoxins). Control of FHB growth and toxin accumulation in grains remain major challenges. While the ultimate goal in the battle against FHB is the development of resistant wheat varieties, the actual use of fully resistant plants that preclude any need for treatment with fungicides remains out of sight. Current antifungals being applied against FHB are generally azole‐based inhibitors. However, usage of these azole‐based fungicides is being complicated by the facts that these are active only during specific short‐lived developmental time periods, fungi are developing increased resistance to them and they are having significant environmental impacts. As such, there is a great need for more targeted, specific and effective antifungal agents to address the significant threat of FHB. This review provides an overview of some of the more promising fungal targets that are currently being investigated for antifungal development.     

Fusarium species and chemotypes associated with fusarium head blight and fusarium root rot on wheat in Sardinia

Environmental conditions in Sardinia (Tyrrhenian Islands) are conducive to fusarium root rot (FRR) and fusarium head blight (FHB). A monitoring survey on wheat was carried out from 2001 to 2013, investigating relations among these diseases and their causal agents. FHB was more frequently encountered in the most recent years while FRR was constantly present throughout the monitored period. By assessing the population composition of the causal agents as well as their genetic chemotypes and EF‐1α polymorphisms, the study examined whether the two diseases could be differentially associated to a species or a population. Fusarium culmorum chemotypes caused both diseases and were detected at different abundances (88% 3‐ADON, 12% NIV). Fusarium graminearum (15‐ADON genetic chemotype) appeared only recently (2013) and in few areas as the causal agent of FHB. In F. culmorum, two haplotypes were identified based on an SNP mutation located 34 bp after the first exon of the EF‐1α partial sequence (60% adenine, 40% thymine); the two populations did not segregate with the chemotype but the A‐haplotype was significantly associated with FRR in the Sardinian data set (P = 0·001), suggesting a possible fitness advantage of the A‐haplotype in the establishment of FRR that was neither dependent on the sampling location nor the sampling year. The SNP determining the Sardinian haplotype is distributed worldwide. The question whether the A‐haplotype segregates with characters facilitating FRR establishment will require further validation on a specifically sampled international data set.     

Species composition and genetic structure of Fusarium graminearum species complex populations affecting the main barley growing regions of South America

Members of the Fusarium graminearum species complex (FGSC), such as F. graminearum and F. asiaticum, are the main cause of fusarium head blight (FHB) of wheat and barley worldwide. In this study, 117 FGSC isolates obtained from commercial barley grain produced in Argentina (n = 43 isolates), Brazil (n = 35), and Uruguay (n = 39) were identified to species and trichothecene genotypes, and analysed using amplified fragment length polymorphism (AFLP) and sequence‐related amplified polymorphism (SRAP) markers. In addition, reductase (RED) and trichothecene 3‐O‐acetyltransferase (Tri101) were sequenced for a subset of 24 isolates. The majority of the isolates (n = 103) were identified as F. graminearum, which was the only species found in Argentina. In Uruguay, only one F. cortaderiae isolate was found among F. graminearum isolates. In Brazil, F. graminearum also dominated the collection (22/35), followed by F. meridionale (8/35), F. asiaticum (2/35), F. cortaderiae (2/35) and F. austroamericanum (1/35). Species were structured by trichothecene genotype: all F. graminearum were of the 15‐acetyldeoxynivalenol (ADON), F. meridionale, F. asiaticum and F. cortaderiae were of the nivalenol (NIV), and F. austroamericanum was of the 3‐ADON genotype. Both AFLP and SRAP data showed high levels of genetic variability, which was higher within than among countries. Isolates were not structured by country of origin. SRAP analysis grouped F. graminearum in a separate cluster from the other species within the complex. However, AFLP analysis failed to resolve the species into distinct clades with partial clustering of F. meridionale, F. austroamericanum, F. asiaticum and F. graminearum isolates.     

Nivalenol‐producing Fusarium cerealis associated with fusarium head blight in winter wheat in Manitoba,Canada

Fusarium head blight (FHB) in small grain cereals is primarily caused by the members of the Fusarium graminearum species complex. These produce mycotoxins in infected grains, primarily deoxynivalenol (DON); acetylated derivatives of DON, 3‐acetyl‐DON (3‐ADON) and 15‐acetyl‐DON (15‐ADON); and nivalenol (NIV). This study reports the isolation of Fusarium cerealis in infected winter wheat heads for the first time in Canada. A phylogenetic analysis based on the TRI101 gene and F. graminearum species‐specific primers revealed two species of Fusarium: F. graminearum sensu stricto (127 isolates) and F. cerealis (five isolates). Chemotype determination based on the TRI3 gene revealed that 65% of the isolates were 3‐ADON, 31% were 15‐ADON and 4% were NIV producers. All the F. cerealis isolates were of NIV chemotype. Fusarium cerealis isolates can often be misidentified as F. graminearum as the morphological characteristics are similar. Although the cultural and macroconidial characteristics of F. graminearum and F. cerealis isolates were similar, the aggressiveness of these isolates on susceptible wheat cultivar Roblin and moderately resistant cultivar Carberry differed significantly. The F. graminearum 3‐ADON isolates were most aggressive, followed by F. graminearum 15‐ADON and F. cerealis NIV isolates. The findings from this study confirm the continuous shift of chemotypes from 15‐ADON to 3‐ADON in North America. In Canada, the presence of NIV is limited to barley samples and the discovery of NIV‐producing F. cerealis species in Canadian wheat fields may pose a serious concern to the Canadian wheat industry in the future.     

Molecular,biological and physiological characterizations of resistance to phenamacril in Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating wheat diseases in China. Phenamacril is a novel cyanoacrylate fungicide with a unique chemical structure and specific mode of action against Fusarium spp. In this study, the molecular, biological and physiological characteristics of laboratory‐induced mutants of F. graminearum with resistance to phenamacril were investigated. Compared to the wildtype strains, the phenamacril‐resistant mutants showed obvious defects in various biological and physiological characteristics, including vegetative growth, carbon source utilization, response to oxidative and osmotic stresses, sensitivity to cell wall and cell membrane integrity inhibitors, cell membrane permeability, glycerol accumulation and pathogenicity. The phenotypes of the phenamacril‐resistant mutants exhibited many variations. Sequencing indicated that the three parental strains studied were identical, and the mutants TXR1, TXR2, BMR1, BMR2, SYR1 and SYR2 each had a single point mutation in the amino acid sequence encoded by the myosin‐5 gene (FGSG_01410). These results provide new reference information for future investigations concerning the resistance mechanism of F. graminearum to phenamacril and could offer important relevant data for the management of FHB caused by F. graminearum.     

Variation in susceptibility to pitch canker disease, caused by Fusarium circinatum, in native stands of Pinus muricata

Two hundred Pinus muricata trees, located within a native forest near Monterey, California, were inoculated with the pitch canker pathogen. Treated branches were removed 10–13 weeks following the initial inoculation, and the lengths of the lesions that developed at each of the inoculation sites were measured. Results indicated that bishop pine exhibits a wide range of variation in susceptibility to pitch canker disease. Of the trees that received only one inoculation, 27% showed almost no lesion development, indicating that they were relatively resistant to the pathogen, while others had significantly longer lesions and thus were more susceptible. Clonal propagation and seed collection from resistant individuals may offer useful strategies for disease management in the future.     

Species composition,toxigenic potential and pathogenicity of Fusarium graminearum species complex isolates from southern Brazilian rice

This study aimed to assess the extent and distribution of Fusarium graminearum species complex (FGSC) diversity in rice seeds produced in southern Brazil. Four species and two trichothecene genotypes were detected among 89 FGSC isolates, based on a multilocus genotyping assay: F. asiaticum (69·6%) with the nivalenol (NIV) genotype, F. graminearum (14·6%) with the 15‐acetyldeoxynivalenol (ADON) genotype, and F. cortaderiae (14·6%) and F. meridionale (1·1%), both with the NIV genotype. Seven selected F. asiaticum isolates from rice produced NIV in rice‐based substrate in vitro, at levels ranging from 4·7 to 84·1 μg g^?1. Similarly, two F. graminearum isolates from rice produced mainly 15‐ADON (c. 15–41 μg g^?1) and a smaller amount of 3‐ADON (c. 6–12 μg g^?1). One F. meridionale and two F. cortaderiae isolates did not produce detectable levels of trichothecenes. Two F. asiaticum isolates from rice and two from wheat (from a previous study), and one F. graminearum isolate from wheat, were pathogenic to both crops at various levels of aggressiveness based on measures of disease severity in wheat spikes and rice kernel infection in a greenhouse assay. Fusarium asiaticum and the reference F. graminearum isolate from wheat produced NIV, and deoxynivalenol and acetylates, respectively, in the kernels of inoculated wheat heads. No trichothecene was produced in kernels from inoculated rice panicles by any of the isolates. These findings constitute the first report of FGSC composition in rice outside Asia, and confirm the dominance of F. asiaticum in rice agroecosystems.     

福建省香蕉枯萎病菌硝酸盐营养突变体的营养体亲和性测定

 Fourteen strains of Fusarium oxysporum f. sp. cubense were induced to produce 146 nitrate-nonutilizing(nit) mutants on a chlorate-containing medium. Among them, there were 117 nit1 mutants(80.14%), 17 nit3 mutants(11.64%) and 12 nitM mutants(8.22%). These strains were divided into two vegetative compatibility groups(VCGs) by the vegetative compatibility tests. Twelve strains of F. oxysporum f. sp. cubense from Musa AAA belonged to VCG1, two trains from Musa ABB belonged to VCG2.